The present invention relates to a novel delivery system for delivering therapeutic agents into living cells, and more particularly, to novel chemical moieties that are designed capable of targeting and/or penetrating cells or other targets of interest and further capable of binding therapeutic agents to be delivered to these cells, and to delivery systems containing same.
All known life forms and life processes are based on proteins which are essential to all biological functions. Consequently, all the illnesses and disorders associated with life involve proteins. Among many other functions, proteins play a key role in signaling pathways of immunological and/or neurological processes and are thus major players in many congenital, chronic and infectious diseases and disorders.
As such, proteins exhibit highly potent therapeutic efficacy. Indeed, proteins have already been used successfully in the treatment of diseases such as cancer, hemophilia, anemia and diabetes.
However, although proteins have enormous therapeutic potential, their widespread use has been limited by several restrictive technical factors. First, proteins remain difficult and expensive to manufacture compared to other pharmaceuticals. Large-scale purification of proteins in bioactive form can be a limiting step in the commercialization of these drugs. Second, many proteins are metabolized or otherwise eliminated rapidly in the body. This results in the need for frequent re-administration, which may also prove to be inefficient when the administered protein fails to reach its intended target due to many delivery related factors. Finally, protein drugs generally must be given by injection which increases the complexity and expense of the treatment, and the disagreeable nature of administration also limits potential clinical applications.
The identification of defective genes responsible for disease states, either through defective control of gene expression which leads to overproduction or underproduction of key proteins, or the production of defective proteins, offers new possibilities for the treatment of disease. By controlling the defect at the genetic level, a range of diseases could potentially be treated effectively rather than by merely treating the symptoms of these diseases.
The use of genetic material to deliver genes for therapeutic purposes has been practiced for many years. Experiments outlining the transfer of DNA into cells of living animals were reported as early as 1950. Later experiments using purified genetic material only further confirmed that the direct DNA gene injection, even in the absence of viral vectors results in the expression of the inoculated genes in the host. There have been additional experiments that extend these findings to recombinant DNA molecules, further illustrating the idea that purified nucleic acids could be directly delivered into a host and proteins would be produced.
Generation of therapeutic gene products (such as polypeptides, proteins, mRNA and RNAi) by expression of therapeutic gene product-encoding DNA in transformed cells has attracted wide attention as a method to treat various mammalian diseases and enhance production of specific proteins or other cellular products. This promising technology, often referred to as gene therapy (Crystal et al., Science 1995, 270, 404 and Rhang et al., Human Gene Therapy, 1999, 10:1735-1737), is generally accomplished by introducing exogenous genetic material into a mammalian patient's cells. Transformed cells can be accomplished by either direct transformation of target cells within the mammalian subject (in vivo gene therapy) or transformation of cells in vitro and subsequent implantation of the transformed cells into the mammalian subject (ex vivo gene therapy) (for reviews, see Chang et al. 1994 Gastroenterol. 106:1076-84; Morsy et al. 1993 JAMA 270:2338-45; and Ledley 1992 J. Pediatr. Gastroenterol. Nutr. 14:328-37). The introduced genetic material can be designed to replace an abnormal (defective) gene of the mammalian patient (“gene replacement therapy”), or can be designed for expression of the encoded protein or other therapeutic product without replacement of any defective gene (“gene augmentation”). Because many congenital and acquired medical disorders result from inadequate production of various gene products, gene therapy provides means to treat these diseases through either transient or stable expression of exogenous nucleic acid encoding the therapeutic product.
Although the initial motivation for gene therapy was the treatment of genetic disorders, it is becoming increasingly apparent that gene therapy will be useful for the treatment of a broader range of acquired diseases such as cancer, infectious disorders (such as AIDS), heart disease, arthritis, and neurodegenerative disorders such as Parkinson's and Alzheimer's diseases.
In addition to gene therapy, other therapeutic approaches at the DNA level are known. These include, for example, gene vaccination and antisense oligonucleotide therapy.
In 1992, scientists Tang and DeVit [Tang, D. C., M. DeVit, et al., 1992, Nature, 356(6365): 152-4] reported that the delivery of human growth hormone in a gene expression cassette in vivo resulted in production of detectable levels of the growth hormone in host mice. They also found that these inoculated mice developed antibodies against the human growth hormone; they termed this immunization procedure “genetic immunization”, which describes the ability of inoculated genes to be individual immunogens. From this seminal work stemmed the concept of gene vaccination, which is based on bacterial expression plasmids. Expression plasmids used in DNA-based vaccination normally contain the antigen expression unit composed of promoter/enhancer sequences, followed by antigen-encoding and polyadenylation sequences and the production unit composed of bacterial sequences necessary for plasmid amplification and selection [Schirmbeck, R. et al., 2001, Biol. Chem., 382:543-552]. The construction of bacterial plasmids with vaccine inserts is accomplished using recombinant DNA technology. Once constructed, mass-produced in bacteria and purified, the DNA acts as the vaccine. More information regarding gene vaccination can be found in many publications such as, for example, by Koprowski, H. and Weiner, D. B., 1998, “DNA Vaccination and Genetic Vaccination”, Spriner-Verlag, Heidelberg, p 198.
The emerging concept of “antisense therapy” focuses on defeating diseases before the proteins which cause them can even be formed. The production of these faulty proteins begins in the cellular DNA. In the nucleus the DNA forms pre-mRNA, which leaves the nucleus to enter the cytoplasm, interacts with the ribosome and translated into the protein. DNA is termed “antisense” when its base sequence is complementary to the gene's messenger RNA, for example a “sense-DNA” segment of 5′-AAGGTC-3′ corresponds to the “antisense-DNA” segment 3′-TTCCAG-5′. While many traditional drugs attempt to defeat the diseases by focusing on the faulty proteins themselves, antisense therapy goes a step further, by preventing the production of these incorrect proteins. The prevention or attenuation of the disease-causing gene expression is accomplished by insertion of the antisense DNA of the disease-producing gene into the cell's cytoplasm, wherein instead of being translated by the ribosome, the disease-producing mRNA hybridizes with the strand of antisense DNA and instead of producing proteins, the faulty mRNA is negated by the antisense oligonucleotide.
DNA is inherently an unstable material in an active biological environment where many specific enzymes capable of degrading and metabolizing DNA are found (Ledoux et al., Prog. Nucl. Acid. Res., 1965, 4, 231). In addition, natural protection against alien DNA exists in the body. Thus, the gene therapy, antisense oligonucleotide therapy and gene vaccination approaches described above require that the DNA and DNA analogues would survive in such a hostile biological environment and in addition, that the DNA and DNA analogs would penetrate biological barriers, be taken up into cells and be delivered to the correct subcellular compartment to exert their therapeutic effects. While some DNA is taken up naturally into cells, the amount taken up is typically small and inconsistent, and expression of added DNA is therefore poor and unpredictable.
A number of strategies have been proposed to achieve delivery of DNA into living cells. These include the use of liposomes (Fraley et al., Proc. Natl. Acad. Sci. USA, 1979, 76, 3348), cationic lipids (Felgner et al., Proc. Natl. Acad. Sci USA, 1987, 84, 7413), and the use of cationic polymers, or polycations, such as polylysine and polyornithine as DNA delivery agents (Farber et al., Biochim. Biophys. Acta, 1975, 390, 298 and Wagner et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 3410).
Unfortunately cationic polymers which have been used for the purpose of DNA delivery were found deficient in a number of respects. Poly-L-lysine, the principal polymer presently used for this purpose, is known to be toxic above a small molecular weight (Clarenc et al., Anticancer Drug Design, 1993, 8, 81), and does not interact stoichiometrically with DNA, leading to an unstable and unreliable complex with DNA.
An alternative for genetic augmentation and therapy using DNA manipulation is the use of RNA molecules, a relatively new concept which has received increasing attention during the past decade. Most genes function by expressing a protein via an intermediate, termed messenger RNA (mRNA), or sense RNA. Therefore, the ability to specifically knock-down expression of a gene of interest, e.g., by complementary mRNA agents, is recognized as powerful tool for regulation of gene expression (Green & Pines, Annu. Rev. Biochem., 1986, 55, 569-597). These complementary RNA molecules, termed antisense RNA molecules, or small interfering RNA (siRNA), specifically recognize their target transcripts (mRNA) by forming base-paired strands with the mRNA in a sequence-dependent manner. The formation of an RNA duplex interferes with the translation of the mRNA into a protein by the ribosome, and further leads to the degradation of the target mRNA by naturally occurring cellular enzymes which target duplex RNA molecules (Hamilton & Baulcombe, Science, 1999, 286:950-952). This phenomenon, also known as gene silencing or RNA interference (RNAi) has been reported to be accompanied by the accumulation of short fragments of double stranded siRNAs, 20-25 nucleotides long, which are reported to be synthesized from an RNA template (Fire et al., Nature, 1998, 391:806-811; Timmons & Fire, Nature, 1998, 395:854; WO99/32619; Kennerdell & Carthew, Cell, 1998, 95:1017-1026; Ngo et al., Proc. Natl. Acad. Sci. USA, 1998, 95:14687-14692; Waterhouse et al., Proc. Natl. Acad. Sci. USA, 1998, 95:13959-13964; WO99/53050; Cogoni & Macino, Nature, 1999, 399:166-169; Lohmann et al., Dev. Biol., 1999, 214:211-214; Sanchez-Alvarado & Newmark, Proc. Natl. Acad. Sci. USA, 1999, 96:5049-5054; and Elbashir et al., Nature, 2001, 411:494-29).
Nevertheless, the use of siRNA for gene silencing also suffers from major drawbacks, which mainly stem from the inherent instability of RNA molecules in a biological environment, and which impede its delivery into cells. Thus, the delivery of intact siRNA molecules into a cell, and more so into the desired cells, is limited by the rapid breakdown of the RNA in the bloodstream, by poor absorption of RNA through the membranes of mammalian cells, and further by the breakdown of the RNA down inside the cell by RNAse enzymes and other scavenger proteins.
In a search for a genetic material delivery platform, researchers have turned their attention to one of nature's most efficient DNA/RNA delivery machines—the viruses. Viruses are known for their ability to be extremely efficient in delivering genes to the particular cells that are required for the survival and progression of the viral species (Smith, Annual. Rev. Microbiol., 1995, 49:807-838). Indeed, studies aimed at understanding the molecular mechanisms in which the viral genetic code is integrated into the cell has paved the path for viral based gene delivery platforms (Wei et al., J. Virol., 1981, 39: 935-944). Yet, an optimal synthetic virus which does not involve serious health-related side effect has not been designed yet.
In order to overcome the obstacle of the rapid and efficient DNA/RNA degradation by scavenging enzymes, one of the impedances on the path to genetic therapy, researches have attempted to generate DNA/RNA derivatives which will be less susceptible to degradation yet still active as a coding sequence, via the manipulation and modification of nucleotides (for example, Draper, Nucleic Acids Res., 1984, 12(2): 989-1002 and Freier and Altmann, Nucleic Acids Res., 1997, 25(22): 4429-43). Yet, these DNA/RNA analogs based on chemically modified nucleotides and nucleotide-mimicking compounds are typically found toxic or otherwise unpredictable and therefore therapeutically unusable, and are mostly used for in vitro research purposes.
Thus, although therapeutic approaches that involve intervention at the gene level are widely recognized as promising technologies, these methods are limited by the absence of an efficient and reliable method of delivering DNA and RNA.
There is thus a widely recognized need for, and it would be highly advantageous to have, a novel delivery system for delivering therapeutic agents such as DNA and RNA molecules into living cells, which would overcome the present limitations associated with gene therapy.